Organelles from squid giant axon move on actin filaments, demonstrating that myosins are likely to be present on their surfaces. In order to understand further the role of myosins in axonal transport it was necessary first to develop an overview of squid myosin structure, biochemical behavior, and sequence. However, myosins proved to be relatively resistant to digestion for microsequencing, and large amounts of protein were needed because digestion of the large heavy chain (200- 230 kD) yielded many peptide fragment of similar sizes which require two purification steps to separate. Because axons yielded too little protein, myosins were purified from syphon muscle and semipurified from optic lobes. The latter myosins were further separated by gel electrophoresis and the band migrating 200-230 kDa excised. Protein was digested in the slice in the presence of SDS, which produced fragments from otherwise protected domains as well as improving the overall yield of all fragments. Adequate amounts of fragments were thus produced from both sources for two successive HPLC separation steps which isolated these peptides sufficiently for Edman degradative sequencing. When the resulting amino acid sequences from optic lobe myosins were mapped against myosin sequences in the data bank, myosin II and myosin V forms predominated. Antibodies raised to the muscle myosin, which also recognize myosins in optic lobes, recognize a high molecular weight, possibly type II, myosin on axonal organelles, while antibodies to chick myosin V recognize a band in the right size range to be myosin V the organelle fraction from the axon. The current goal is to characterize the higher molecular weight myosin recognized by the muscle myosin antibody because of its likely role in some aspects of axonal transport.